This invention relates to a process for producing ethanol. More particularly, it relates to a process for producing ethanol from a D-sugar.
Yeasts of the genus Saccharomyces have been found to be generally good alcoholic fermentors, capable of producing ethanol from a variety of naturally occurring D-sugars. Saccharomyce cerevisiae is more commonly used for industrial alcohol production. Usual growth requirements for the yeast include a carbon and energy source, which is supplied by the sugar, a nitrogen source which may be supplied as ammonium salts, urea or peptone, and a source of certain vitamins (particularly those of the B-complex), which, if not present in some naturally occurring crude feedstock, can be supplied in the form of a yeast extract. Also required is a mineral source, which, if not present in the naturally occurring feedstock, or water, can be supplied in the form of a yeast extract.
Yeast will generally produce ethanol under two environmental conditions. These conditions are (a) a lack of oxygen (i.e., oxygen-limited conditions) and (b) the presence of a high sugar content. In producing ethanol from D-sugars, there is a need to produce the ethanol in a continuous culture. The continuous culture as yeast cell mandates the continuous propagation of cells. The yeast cell concentration is at a steady state when the rate of cell growth equals the rate at which cells are drained off or die. The cell dilution rate is proportional to the feed-in rate and is equal to the specific growth rate of the organism, i.e., the yeast mutant, in the steady state.
In an industrial continuous fermentation process for the production of ethanol, the sugar concentration in the fermentor effluent should be near zero. However, a continuous fermentation cannot be conducted under completely anaerobic conditions, since oxygen is needed by the yeast as an essential nutrient for growth.
The oxygen control is critical in a continuous ethanol culture especially when using a single fermentor. Too much oxygen results in mitochondrial biogenesis, which leads to oxidative enzyme formation and thus to more yeast cells at the expense of ethanol production. Too little oxygen causes cessation of growth and, therefore, total loss of the continuous culture.
Continuous fermentation to produce ethanol can be conducted with the addition of a small controlled amount of oxygen to the continuous fermentor, with the purpose of keeping the yeast in an active state. This was shown in the study of Cysewski, G. R. and Wilke, C. R., Biotechnol. Bioeng. 20 1421 (1978).
In continuous fermentation of D-sugars to ethanol, a high rate of ethanol production per unit of fermentor volume is desirable to limit fermentor size and cost. Fermentors can be made more productive by recycling yeast cells. Ease of separation of the yeast cells from the fermentor effluent is highly desirable to effect this recycle of yeast cells. Traditional strains of Saccharomyce cerevisiae are difficult to separate from the fermentor effluent and require the use of expensive centrifugation and filtration equipment. Even the centrifuged yeast has a considerable amount of liquids adhering, which makes the recycle less efficient due to the fact that the recycled ethanol reduces the fermentation rate.
Flocculating strains of the Saccharomyces yeast have been developed, which are easier to separate from the fermentor effluent and do not require costly contrifuges.
Respiration-deficient yeast mutants (RDM), also called Rho mutants as described by Aiba, S., Shoda, M., and Nagatine, M., in Biotechnol. Bioeng. 10 845 (1968), can ferment glucose to ethanol in the presence of excess oxygen. This has the advantage that oxygen addition need not be controlled precisely and thus alleviates many process control problems. Under normal conditions, in which sufficient aeration is provided, insufficient ethanol production results due to the well documented Pasteur effect, which occurs when glucose is metabolized in a series of reactions which by-pass ethanol production in order to derive the energy needed by the cell. This reaction sequence involves a number of enzymes and is called respiration, since oxygen serves as the terminal electron acceptor in the series of events. In order to avoid the loss of productivity associated with this process, a mutation can be induced in the yeast, which results in the loss of respiratory activity. Such mutants are prepared by a standard procedure first described by Boris Euphrussi [Unites Biologiques Dourees de Continuite Genetique, Paris, June-July, pp. 165-180, Editions du C.M.R.S., Paris (1949)] using acriflavin dye.
This mutation, known as petite due to its effect on colony size, is very stable and allows for ethanol production in an aerobic environment so that the Pasteur effect is avoided.
Aiba, et al demonstrated a rate of ethanol production by a respiration deficient mutant of Saccharomyce cerevisiae of only 1.69 grams per liter of fermentor volume per hour. This activity of the Rho mutant was inhibited by an ethanol concentration of 1%. Therefore, the feed glucose concentration could not exceed 20 grams per liter and the ethanol yield could not exceed 10 grams per liter or 1%, which would be difficult if the process were expanded to an industrial scale. Furthermore, the cell dry mass obtained in this system was only 2.3 grams per liter.
The above study tends to indicate that respiration-deficient mutants (RDM) cannot be advantageously used to industrially produce ethanol from a D-sugar. However, as discussed below, by the present invention it was found that a respiration-deficient mutant (RDM) strain of Saccharomyces uvarum tends to produce a a high yield of ethanol in the presence of air.